Engineered TGF-β Monomers and Methods of Use

Abstract

Recombinant TGF-β2 monomers engineered to prevent dimerization and block TGF-β signaling are described. The engineered monomers lack the ability to bind and recruit TGF-β type I receptor (TβRI) but retain the ability to bind high affinity TGF-β type II receptor (TβRII). The TGF-β2 monomers also contain additional modifications that increase their affinity for TβRII, reduce their aggregation, and / or improve their folding. Nucleic acid molecules and vectors encoding recombinant TGF-β2 monomers are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 254,249, filed October 11, 2021, which is incorporated by reference in its entirety herein.

[0002] Field The present disclosure relates to transforming growth factor (TGF)-β monomers with improved properties and their use for inhibiting TGF-β signaling and treating disorders associated with aberrant TGF-β signaling.

[0003] Acknowledgement of Government Support This invention was made with Government support under Grant No. CA247129 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] background TGF-β is an important target for cancer immunotherapy because TGF-β-mediated immunosuppression is mediated by the regulatory T (T reg) cells suppress antitumor immunity through secretion of immunosuppressive cytokines and direct inhibition of effector T cells, resulting in an insufficient cytotoxic environment. Overexpression of TGF-β in the tumor microenvironment (TME) correlated with high tumor burden and poor clinical outcomes. Furthermore, checkpoint therapy non-responders have a T cell exhausted phenotype due to TGF-β-mediated immune exclusion. TGF-β inhibitors have further been shown to transcend checkpoint monotherapy in vivo as an adjunct to PD-1 and PD-L1 therapy, and such approaches are being pursued in clinical trials. TGF-β isoforms can also potently stimulate accumulation of matrix proteins (e.g., collagen and fibronectin) and drive fibrotic disorders (e.g., idiopathic pulmonary fibrosis (IPF), renal fibrosis, cardiac fibrosis, and coronary restenosis). IPF, which is characterized by progressive loss of lung function occurring in older adults, has a median survival rate of 5 years after diagnosis. Although the mechanisms inducing different forms of fibrosis are diverse, they share a common induction of increased levels of TGF-β protein. In IPF and renal fibrosis, increased levels of TGF-β protein stimulate the activation and differentiation of fibroblasts into myofibroblasts, which causes abnormal deposition of extracellular matrix (ECM), resulting in scarring and impaired organ function.

[0005] Inhibition of TGF-β in both cancer and fibrosis contexts using neutralizing antibodies has limited efficacy, since most of TGF-β is likely stored as latent protein in the ECM, making it difficult to inhibit. TGF-β receptor kinase inhibitors make their target, the kinase domain of TGF-β type I and type II receptors, more accessible, but lack specificity and inhibit not only other TGF-β family type I receptors, but also non-TGF-β receptor kinases. Kinase inhibitors have not progressed beyond phase II in clinical trials, and currently there are no FDA-approved TGF-β inhibitors. Thus, improved therapies are needed to treat disorders associated with abnormal TGF-β signaling. Summary of the Invention [Means for solving the problem]

[0006] overview Described herein is a TGF-β2 monomer engineered to prevent dimerization and block TGF-β signaling. The engineered monomer lacks the ability to bind and recruit TGF-β type I receptor (TβRI), but retains the ability to bind high affinity TGF-β type II receptor (TβRII). The TGF-β2 monomers of the present disclosure also contain additional modifications that increase their affinity to TβRII, reduce their aggregation, and / or improve their folding. The TGF-β2 monomers and compositions thereof of the present disclosure can be used, for example, to treat disorders associated with abnormal TGF-β signaling (e.g., fibrotic disorders and cancer).

[0007] Provided herein is a recombinant TGF-β2 monomer that includes a deletion of the α3 (heel) helix corresponding to amino acid residues 52-71 of wild-type human TGF-β2 (shown as SEQ ID NO:1), and a cysteine ​​to arginine or serine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1; these modifications prevent dimerization of the monomer. The TGF-β2 monomer further includes a leucine to arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO:1, and an alanine to lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO:1; these modifications increase the net charge of the monomer. The TGF-β2 monomer also includes a lysine to arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO:1, and a lysine to arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO:1, which increases the affinity of the monomer for TβRII. In some implementations, the TGF-β2 monomer further comprises one or more additional modifications that increase affinity of the monomer for TβRII, reduce aggregation, and / or improve folding.

[0008] Also provided herein is an engineered TGF-β2 monomer that has been modified to contain the cystine-knot region of Dan and Cerberus related protein (PRDC), which enhances folding of the monomer.

[0009] Also provided is a fusion protein comprising a TGF-β2 monomer and a heterologous protein. In some implementations, the heterologous protein includes a protein tag, an Fc domain, albumin, an albumin-binding polypeptide, an antibody, an antigen-binding fragment of an antibody, or a targeting moiety.

[0010] Nucleic acid molecules and vectors encoding the recombinant TGF-β2 monomer or fusion proteins disclosed herein are also provided. Further provided are isolated cells (e.g., isolated T cells) comprising a nucleic acid molecule or vector encoding a recombinant TGF-β2 monomer or fusion protein.

[0011] Further provided is a composition comprising a recombinant TGF-β2 monomer, fusion protein, nucleic acid molecule, vector, or isolated cell disclosed herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0012] Also provided are methods of inhibiting TGF-β signaling in a cell by contacting the cell with a recombinant TGF-β2 monomer, fusion protein, nucleic acid molecule, vector, or composition disclosed herein.

[0013] Further provided is a method of inhibiting TGF-β signaling in a subject having a disease or disorder associated with abnormal TGF-β signaling. In some implementations, the method comprises administering to the subject an effective amount of a recombinant TGF-β2 monomer, a fusion protein, a nucleic acid molecule, a vector, an isolated cell (e.g., T cell) or a composition disclosed herein. Further provided is a method of treating a disease or disorder associated with abnormal TGF-β signaling in a subject. In some implementations, the method comprises administering to the subject a therapeutically effective amount of a recombinant TGF-β2 monomer, a fusion protein, a nucleic acid molecule, a vector, an isolated cell (e.g., T cell) or a composition disclosed herein. In some examples of the methods of the present disclosure, the disease or disorder associated with abnormal TGF-β signaling is a fibrotic disorder, a cancer, an eye disorder or a genetic disorder of connective tissue.

[0014] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0015] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1-1]1A-1E: Sequence comparison of engineered TGF-β2 monomers mmTGF-β2-7M (SEQ ID NO: 3) (FIG. 1A), mmTGF-β2-7M2R (SEQ ID NO: 4) (FIG. 1B), mmTGF-β2-2M-Del7_16 (SEQ ID NO: 5) (FIG. 1C), mmTGF-β2-7M-PRDC (SEQ ID NO: 7) (FIG. 1D), and mmTGF-β2-7M2R-Del7-16 (SEQ ID NO: 6) (FIG. 1E) to TGF-β2 (SEQ ID NO: 1). Sequence differences are indicated by numbers below the two aligned sequences, indicating the nature of the difference. Sequence identity is indicated by an asterisk. Shown below the sequences in Figure 1A are the structures of the TGF-β3-(TβRII)2-(TβRI)2 complex (PDB 2PJY) (left) and the mmTGF-β2-7M-TβRII complex (PDB 5TX4) (right), with some of the main structural features highlighted. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above.

[0016] [Diagram 2] Figures 2A-2F: Amide 1H-15N one-bond shift correlation nuclear magnetic resonance (NMR) spectra of mmTGF-β2-7M2R (Figures 2A-2C) compared to the parent protein, mmTGF-β2-7M (Figures 2D-2F). Spectra were recorded at 37°C in 10 mM phosphate buffer at pH 4.6 (Figures 2A and 2D) or pH 7.2, either in the absence of CHAPS in the buffer (Figures 2B and 2E) or with CHAPS added to a final concentration of 10 mM (Figures 2C and 2F).

[0017] [Diagram 3]Figures 3A-3C: Amide 1H-15N 1-bond shift correlation NMR spectra of mmTGF-β2-2M-Del7-16. Spectra were recorded at 37°C in 10 mM phosphate buffer, pH 6.0 (Figure 3B), or pH 4.5, either in the absence of CHAPS in the buffer (Figure 3A) or with CHAPS added to a final concentration of 10 mM (Figure 3C).

[0018] [Figure 4] Figures 4A-4D: Amide 1H-15N 1 bond shift correlation NMR spectra of mmTGF-β2-7M-PRDC (Figures 4A-4C) and binding to TβRII as detected by native gel electrophoresis (Figure 4D). Spectra were recorded at 37 °C in 10 mM phosphate buffer at pH 4.8 (Figure 4A) or pH 6.0 (Figures 4B-4C). Figures 4B and 4C differ only in the contour level at which the signal is plotted (Figure 4B is plotted at a contour level closer to the noise compared to panel Figure 4C). The native gels shown in Figure 7D were performed by running either 2 μg of TβRII alone (leftmost lane) or the engineered TGF-β monomers mentioned above added at specific molar ratios (+A and +B indicate TβRII:engineered TGF-β monomers at either a 1:1 or 2:1 molar ratio, respectively).

[0019] [Diagram 5] FIG. 5: Binding of the engineered TGF-β monomers (mmTGF-β2-7M, left; mmTGF-β2-7M2R, center; and mmTGF-β2-2M-Del7-16, right) to the TGF-β type II receptor (TβRII) as detected by isothermal titration calorimetry (ITC). The upper panel shows the raw thermograms for three replicate titrations, while the lower panel shows the integrated heats (data points) for three replicate titrations globally fitted to a 1:1 binding isotherm (smooth line). The fitted parameters are provided in the table below.

[0020] [Figure 6]Figures 6A-6D: HEK-293 cell-based CAGA-Luc TGF-β reporter assay evaluating the inhibitory potency of the engineered TGF-β monomers relative to each other. HEK-293 cells stably transfected with a TGF-β CAGA-Luc reporter were treated with the indicated engineered TGF-β monomers at the specified concentrations for 30 minutes and then stimulated by the addition of 10 pM TGF-β3. Cells were harvested 14 hours later and assayed for luciferase activity. (Figure 6A) mmTGF-β2-7M (SEQ ID NO: 3), IC50 of 58.23 nM. (Figure 6B) mmTGF-β2-7M2R (SEQ ID NO: 4), IC50 of 53.29 nM. (Figure 6C) mmTGF-β2-2M-Del7-16 (SEQ ID NO: 5), IC50 of 111.0 nM. (FIG. 6D) mmTGF-β2-7M-PRDC (SEQ ID NO: 7), IC50 is 282.5 nM. Data points and error bars shown represent the mean and standard deviation of triplicate determinations. The smooth curve represents a fit to a standard dose-response inhibition isotherm. The fitted IC50 value is shown.

[0021] Sequence Listing The sequence listing has been submitted as an ST.26 Sequence Listing XML file entitled 8123-107062-03 (created on October 3, 2022, and is 7561 bytes in size), which is incorporated herein by reference. In the attached sequence listing:

[0022] SEQ ID NO:1 is the amino acid sequence of wild-type human TGF-β2.

[0023] SEQ ID NO:2 is the amino acid sequence of an engineered human TGF-β2 monomer, designated mmTGF-β2.

[0024] SEQ ID NO:3 is the amino acid sequence of an engineered human TGF-β2 monomer designated mmTGF-β2-7M.

[0025] SEQ ID NO:4 is the amino acid sequence of an engineered human TGF-β2 monomer designated mmTGF-β2-7M2R.

[0026] SEQ ID NO:5 is the amino acid sequence of an engineered human TGF-β2 monomer designated mmTGF-β2-2M-Del7-16.

[0027] SEQ ID NO:6 is the amino acid sequence of an engineered human TGF-β2 monomer designated mmTGF-β2-7M2R-Del7-16.

[0028] SEQ ID NO:7 is the amino acid sequence of an engineered human TGF-β2 monomer designated mmTGF-β2-7M-PRDC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description I. Abbreviations CKGF Cystine knot growth factor fold ECM Non-cellular matrix ER endoplasmic reticulum GFD Growth factor domain Idiopathic pulmonary fibrosis (IPF) ITC Isothermal Titration Calorimetry NGF Nerve Growth Factor NMR nuclear magnetic resonance PDGF Platelet-derived growth factor Proteins related to PRDC Dan and Cerberus TGF-β Transforming Growth Factor β TβRI Transforming growth factor-β type I receptor TβRII Transforming growth factor-β type II receptor TME Tumor Microenvironment VEGF Vascular endothelial growth factor

[0030] II. Terminology Unless otherwise noted, technical terms are used according to conventional usage.Definitions of common terms in the field of molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 2008, Vol. 16; and other similar references.

[0031] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes a protein or a plurality of antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It should be further understood that any and all base or amino acid sizes, and all molecular weight or molecular mass values, when given for nucleic acids or polypeptides, are approximate and are provided for illustrative purposes unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will take precedence. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. To facilitate scrutiny of various implementations, the following explanations of terms are provided:

[0032] Aberrant (TGF-β signaling): Aberrant or dysregulated TGF-β signaling. In the context of this disclosure, "aberrant TGF-β signaling" refers to excessive (pathological) activation of the TGF-β signaling pathway.

[0033] Administration: Providing or giving an agent (e.g., a therapeutic agent (e.g., a TGF-β monomer)) to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection or infusion (e.g., intratumoral, subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraprostatic, intraventricular, intrastriatal, intracranial and spinal), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0034] Contacting: To place into direct physical association; includes both solid and liquid forms. When used in the context of in vivo methods, "contacting" also includes administering.

[0035] Fibrosis: The formation of excess fibrous connective tissue in an organ or tissue during repair or reaction processes. Fibrosis can occur in many different body tissues (e.g., heart, lung and liver), typically as a result of inflammation or injury. Fibrotic disorders include, but are not limited to, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, liver cirrhosis, renal fibrosis (e.g., from damage caused by diabetes), atrial fibrosis, endomyocardial fibrosis, atherosclerosis, restenosis and scleroderma. Fibrosis can also occur as a result of surgery complications, chemotherapy drugs, radiation, injury or burns.

[0036] Fusion protein: A protein that contains at least portions of two different (heterologous) proteins. In some implementations herein, the fusion protein comprises a TGF-β2 monomer fused to a protein tag, an Fc domain (e.g., a human Fc domain), or albumin.

[0037] Glycosylation: The process of covalent attachment of carbohydrate moieties to asparagine (N-glycosylation), or serine or threonine residues (O-glycosylation). The level and type of glycosylation may vary between different host organisms used for recombinant expression. Novel glycosylation sites may be engineered into sequences by introducing glycosylation sequons into solvent-exposed regions of a protein. For example, the N-glycosylation sequon, NX[S / T], may be introduced at one or more locations within the sequence of a particular implementation disclosed herein. Variation of the type and extent of glycosylation has practical applications in modulating solubility, function and half-life, as well as enabling site-specific chemical conjugation.

[0038] Heterologous: Derived from a distinct genetic source or species.

[0039] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein (including an antibody), organelle, or recombinant virus) has been substantially separated or purified away from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., a cell) in which it is present. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. Isolated does not require absolute purity and can include proteins, peptides, nucleic acid molecules, or viruses that are at least 50% isolated (e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated).

[0040] Modification: A change in the sequence of a nucleic acid sequence or a protein sequence. For example, amino acid sequence modifications include, for example, substitutions, insertions and deletions, or combinations thereof. Insertions include amino- and / or carboxyl-terminal fusions, as well as intrasequence insertions of single or multiple amino acid residues. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. In some implementations herein, the modification (e.g., substitution, insertion, or deletion) results in a change in function (e.g., a reduction or enhancement of a particular protein activity (e.g., reduced aggregation, improved folding, or increased affinity for a target protein)). A substantial modification is one in which at least one residue has been removed and a different residue has been inserted in its place. Amino acid substitutions are typically of a single residue, but can occur at many different positions at once. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at a final mutant sequence. These modifications can be prepared by modification of nucleotides in the DNA encoding the protein, thereby generating DNA encoding the modifications. Techniques for making insertion, deletion and substitution mutations at predetermined sites in DNA having a known sequence are known. A "modified" protein or nucleic acid is one that has one or more of the modifications as outlined above.

[0041] Monomer: A single molecular unit (e.g., a protein) that can bind to other molecular units to form dimers or polymers. In the context of the present disclosure, a "TGF-β2 monomer" is a single TGF-β2 polypeptide chain, the wild-type version of which can bind other TGF-β2 monomers to form dimers. In some implementations herein, the recombinant TGF-β2 monomers are engineered to prevent dimerization. In other implementations herein, the recombinant TGF-β2 monomers that are engineered to prevent their direct dimerization can be fused to a heterologous protein (e.g., the Fc domain of IgG) that can dimerize with itself.

[0042] Neoplasm, Malignancy, Cancer or Tumor: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can give rise to a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of the tumors. Tumors that do not metastasize are said to be "benign." Tumors that can invade surrounding tissues and / or metastasize are said to be "malignant."

[0043] Examples of hematological neoplasms include leukemias (acute leukemias (e.g., 11q23 positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0044] Examples of solid tumors (e.g., sarcomas and carcinomas) include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer (including basal, ductal, and lobular breast cancer), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, and pheochromocytoma. These include sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical carcinoma, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0045] PEGylation: The process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures (e.g., drugs, therapeutic proteins, or vesicles), which are then said to be PEGylated (or pegylated). PEGylation is conventionally achieved by incubating a reactive derivative of PEG with the target molecule. Covalent attachment of PEG to a drug or therapeutic protein can hide the agent from the host's immune system (reducing immunogenicity and antigenicity) and increase the hydrodynamic size (size in solution) of the agent, which extends its circulation time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

[0046] Peptide or Polypeptide: A polymer in which the monomers linked together through amide bonds are amino acid residues. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer may be used. The terms "peptide", "polypeptide", or "protein", as used herein, are intended to encompass any amino acid sequence, including modified sequences. The terms "peptide" and "polypeptide" are specifically intended to encompass naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

[0047] Conservative amino acid substitutions are those substitutions that, when made, minimally interfere with the properties of the original protein, i.e., the structure and especially the function of the protein are preserved and not significantly altered by such substitutions. Examples of conservative substitutions are shown below. [Table 2-1] [Table 2-2]

[0048] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone (e.g., as a sheet or helix conformation) in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0049] Substitutions that are generally predicted to result in the largest changes in protein properties are non-conservative, for example, (a) a hydrophilic residue (e.g., serine or threonine) is replaced with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) a cysteine ​​or proline is replaced with any other residue; (c) a residue with a positively charged side chain (e.g., lysine, arginine, or histidine) is replaced with a negatively charged residue (e.g., glutamic acid or aspartic acid); or (d) a residue with a bulky side chain (e.g., phenylalanine) is replaced with one without a side chain (e.g., glycine).

[0050] Pharmaceutically acceptable carriers: The pharma- ceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington: The Science and Practice of Pharmacy, edited by The University of the Sciences in Philadelphia, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents (e.g., recombinant TGF-β2 monomer).

[0051] Generally, the nature of the carrier depends on the particular mode of administration to be used.For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as vehicle).For solid compositions (e.g., powder, pill, tablet or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate).

[0052] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or a pathological condition after it has developed (e.g., reducing the tumor burden (e.g., reducing the volume or size of a tumor) or reducing the number or size of metastases). "Ameliorating" refers to a reduction in the number or severity of a sign or symptom of a disease.

[0053] Recombinant: A recombinant nucleic acid, protein, or virus is one that has a sequence that does not occur in nature or that is created by artificially combining two separate segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques. The term recombinant includes nucleic acids, proteins, and viruses that have been altered by the addition, substitution, or deletion of portions of a naturally occurring nucleic acid molecule or protein.

[0054] Sequence identity / similarity: Identity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more significant when the ortholog proteins or cDNAs are derived from more closely related species (e.g., human and mouse sequences) compared to more distantly related species (e.g., human and C.elegans sequences).

[0055] Methods for alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0056] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Further information can be found at the NCBI website.

[0057] Subject: Living multi-cellular organisms, including vertebrates (a category that includes both human and non-human mammals).

[0058] Tag: A molecule that can be attached to a protein or nucleic acid (e.g., for labeling, detection, or purification purposes). In some implementations, the tag is a protein tag. In some implementations, the protein tag is an affinity tag (e.g., Avitag, hexahistidine, chitin-binding protein, maltose-binding protein, or glutathione-S-transferase), an epitope tag (e.g., V5, c-myc, HA, or FLAG), or a fluorescent tag (e.g., GFP or another well-known fluorescent protein).

[0059] Therapeutically effective amount: A sufficient amount of a compound or composition, e.g., recombinant TGF-β2 monomer, to achieve a desired effect in a subject being treated. For example, this may be the amount necessary to inhibit or block TGF-β signaling in a cell. In other cases, this may be the amount necessary to inhibit or suppress the growth of a tumor. In one implementation, a therapeutically effective amount is the amount necessary to eliminate, reduce the size of, or prevent the metastasis of a tumor (e.g., reduce tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and / or reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%), for example, compared to the size / volume / number before treatment. In one implementation, the therapeutically effective amount is the amount required to increase the survival time of a subject, for example, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, or at least 5 years, compared to the survival time of a subject with the same cancer without treatment with the recombinant TGF-β2 monomer. In other cases, the therapeutically effective amount is the effect required to inhibit or reduce fibrosis, for example, to reduce fibrosis by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100% compared to before treatment. When administered to a subject, a dosage that achieves a target tissue concentration (e.g., in a tumor) that has been shown to achieve the desired in vitro effect is generally used.

[0060] Transforming growth factor-β (TGF-β): A secreted, multifunctional protein that regulates proliferation, cell differentiation, and many other cellular functions. Many cells synthesize TGF-β, and nearly all cells express receptors for TGF-β. The term "TGF-β" refers to three distinct protein isoforms, TGF-β1, TGF-β2, and TGF-β3, encoded by the genes TGFB1, TGFB2, and TGFB3, respectively.

[0061] TGF-β signaling pathway: A signaling pathway involved in many cellular processes, such as cell proliferation, differentiation and apoptosis. Members of the TGF-β pathway include, but are not limited to, TGF-β1, TGF-β2, TGF-β3, TGF-β receptor type I and TGF-β receptor type II.

[0062] TGF-β receptor: The term "TGF-β receptor" includes TGF-β receptor type I (TβRI, encoded by TGFBR1) and TGF-β receptor type II (TβRII, encoded by TGFBR2). TGF-β receptors are serine / threonine protein kinases. When bound to TGF-β, the above-mentioned type I and type II TGF-β receptors form a heterodimer complex and transmit the TGF-β signal from the cell surface to the cytoplasm.

[0063] III. Recombinant TGF-β2 Monomer Disclosed herein is a TGF-β2 monomer engineered to prevent dimerization and block TGF-β signaling. The engineered monomer lacks the ability to bind and recruit TGF-β type I receptor (TβRI), but retains the ability to bind high affinity TGF-β type II receptor (TβRII). The TGF-β2 monomers of the present disclosure also contain additional modifications that increase their affinity for TβRII, reduce their aggregation, and / or improve their folding. The TGF-β2 monomers and compositions thereof of the present disclosure can be used, for example, to inhibit TGF-β signaling in cells or subjects, or to treat disorders associated with abnormal TGF-β signaling (e.g., fibrotic disorders, cancer, eye diseases, or genetic disorders of connective tissue).

[0064] Provided herein is a recombinant TGF-β2 monomer that includes a deletion of the α3 helix corresponding to amino acid residues 52-71 of wild-type human TGF-β2 (shown as SEQ ID NO:1) and a cysteine ​​to arginine (or serine) substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1; these modifications prevent dimerization of the monomer. The TGF-β2 monomer further includes a leucine to arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO:1, and an alanine to lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO:1; these modifications increase the net charge of the monomer. The TGF-β2 monomer also includes a lysine to arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO:1, and a lysine to arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO:1, which enhances affinity for TβRII. The TGF-β2 monomer optionally further includes one or more additional modifications that increase affinity of the monomer for TβRII, reduce aggregation, and / or improve folding.

[0065] In some implementations, the TGF-β2 monomer further comprises an arginine to lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO:1; a valine to arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO:1; a leucine to valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO:1; an isoleucine to valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO:1; a threonine to lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO:1; and an isoleucine to valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO:1. In some examples, the TGF-β2 monomer has a cysteine ​​to arginine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1. In certain examples, the amino acid sequence of the TGF-β2 monomer is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4 (while retaining the amino acid substitutions listed above). In a specific, non-limiting example, the amino acid sequence of said TGF-β2 monomer comprises or consists of the amino acid sequence of mmTGF-β2-7M2R, set forth herein as SEQ ID NO:4.

[0066] In other implementations, the TGF-β2 monomer further comprises a cysteine ​​to valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO:1; and a cysteine ​​to alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO:1. In some examples, the TGF-β2 monomer has a cysteine ​​to serine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1. In particular examples, the amino acid sequence of the TGF-β2 monomer is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5 (while retaining the amino acid substitutions listed above). In specific non-limiting examples, the amino acid sequence of the TGF-β2 monomer comprises or consists of the amino acid sequence of mmTGF-β2-2M-Del7-16, set forth herein as SEQ ID NO:5.

[0067] In other implementations, the TGF-β2 monomer further comprises a cysteine ​​to valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO:1; a cysteine ​​to alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO:1; an arginine to lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO:1; a valine to arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO:1; a leucine to valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO:1; an isoleucine to valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO:1; a threonine to lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO:1; and an isoleucine to valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO:1. In some examples, the TGF-β2 monomer has a cysteine ​​to arginine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1. In particular examples, the amino acid sequence of the TGF-β2 monomer is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical (while retaining the amino acid substitutions recited above) to SEQ ID NO: 6. In specific non-limiting examples, the amino acid sequence of the TGF-β2 monomer comprises or consists of the amino acid sequence of mmTGF-β2-7M2R-Del7-16 ("variant 1" or "var1"), set forth herein as SEQ ID NO: 6.

[0068] Also provided herein is a recombinant TGF-β2 monomer that has been modified to include the cystine-knot region of Dan and Cerberus-related protein (PRDC) to enhance folding of the monomer. In some implementations, the amino acid sequence of the TGF-β2 monomer is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In certain examples, the amino acid sequence of the TGF-β2 monomer comprises or consists of the amino acid sequence of mmTGF-β2-7M-PRDC, set forth herein as SEQ ID NO: 7.

[0069] In some implementations herein, the recombinant TGF-β2 monomer is PEGylated, glycosylated, hyperglycosylated, or includes another modification that enhances circulation time.

[0070] In some implementations, the recombinant TGF-β2 monomer further comprises a radiotherapeutic agent, a cytotoxic agent for chemotherapy, a drug, an imaging agent, a fluorescent dye, or a fluorescent protein tag.

[0071] Also provided herein is a fusion protein comprising TGF-β2 monomer and a heterologous protein.In some implementations, the heterologous protein is a protein tag.In some examples, the protein tag is an affinity tag (e.g., Avitag, hexahistidine, chitin-binding protein, maltose-binding protein, or glutathione-S-transferase), an epitope tag (e.g., V5, c-myc, HA, or FLAG), or a fluorescent tag (e.g., GFP or another well-known fluorescent protein).

[0072] In other implementations, the heterologous protein comprises an Fc domain (e.g., a mouse or human Fc domain). In specific implementations, the heterologous protein promotes intermolecular association of the fusion protein into a homodimeric state (e.g., Fc domain from human IgG1, IgG2, IgG3), a heterodimeric state (e.g., engineered Fc domain, E / K coiled coil), or a multimeric state (e.g., pentabody, nanoparticle). Thus, in some examples, the fusion protein is a single chain polypeptide. In other examples, the fusion protein forms a dimeric or multimeric polypeptide. In specific examples, the fusion protein is a heterodimer.

[0073] In other implementations, the heterologous protein is albumin, an albumin binding protein or agent, or another protein that increases the circulation time of the TGF-β monomer in vivo.

[0074] Further provided herein is an isolated nucleic acid molecule encoding the recombinant TGF-β2 monomer or fusion protein disclosed herein. In some implementations, the nucleic acid molecule is operably linked to a promoter, for example, a T cell specific promoter.

[0075] Also provided is a vector comprising the disclosed nucleic acid molecule.In some examples, the vector is a viral vector (e.g., lentiviral vector).Further provided is an isolated cell comprising the disclosed nucleic acid molecule or vector.In some examples, the cell is a T cell.The cells can be autologous to the subject, or they can be xenogeneic (allogeneic).

[0076] Further provided herein are compositions comprising a recombinant TGF-β2 monomer, fusion protein, nucleic acid molecule, vector, or isolated cell disclosed herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0077] Also provided herein is a method of inhibiting TGF-β signaling in a cell. In some implementations, the method comprises contacting the cell with an effective amount of a recombinant TGF-β2 monomer, fusion protein, nucleic acid molecule, vector, isolated cell or composition disclosed herein. In some examples, the method is an in vitro method. In other examples, the method is an ex vivo method. In yet another example, the method is an in vitro method.

[0078] Further provided is a method of inhibiting TGF-β signaling in a subject having a disease or disorder associated with abnormal TGF-β signaling. In some implementations, the method comprises administering to the subject an effective amount of a recombinant TGF-β2 monomer, fusion protein, nucleic acid molecule, vector, isolated cell or composition disclosed herein. In some examples, the disease or disorder associated with abnormal TGF-β signaling is a fibrotic disorder (e.g., pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, liver cirrhosis, renal fibrosis (e.g., resulting from damage caused by diabetes), atrial fibrosis, endomyocardial fibrosis, atherosclerosis, restenosis, scleroderma, or fibrosis caused by surgery complications, chemotherapy drugs, radiation, injury or burn). In another example, the disease or disorder associated with abnormal TGF-β signaling is breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, renal cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer (e.g., malignant melanoma), or thyroid cancer. In another example, the disease or disorder associated with abnormal TGF-β signaling is an eye disease. In yet another example, the disease or disorder associated with abnormal TGF-β signaling is a genetic disorder of connective tissue.

[0079] Further provided is a method for treating a disease or disorder associated with abnormal TGF-β signaling in a subject. In some implementations, the method comprises administering to the subject a therapeutically effective amount of a recombinant TGF-β2 monomer, fusion protein, nucleic acid molecule, vector, isolated cell or composition disclosed herein. In some examples, the disease or disorder associated with abnormal TGF-β signaling is a fibrotic disorder (e.g., pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, liver cirrhosis, renal fibrosis (e.g., resulting from damage caused by diabetes), atrial fibrosis, endomyocardial fibrosis, atherosclerosis, restenosis, scleroderma, or fibrosis caused by complications of surgery, chemotherapy, radiation, injury or burn). In another example, the disease or disorder associated with abnormal TGF-β signaling is breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, renal cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer (e.g., malignant melanoma), or thyroid cancer. In another example, the disease or disorder associated with abnormal TGF-β signaling is an eye disease. In yet another example, the disease or disorder associated with abnormal TGF-β signaling is a genetic disorder of connective tissue.

[0080] IV. Administration of engineered TGF-β monomers Provided herein is a composition (e.g., a pharmaceutical composition) comprising a recombinant human TGF-β2 monomer, a fusion protein, or a nucleic acid molecule or vector encoding a TGF-β2 monomer or a fusion protein.Also provided is a composition comprising an isolated cell (e.g., a T cell) comprising a vector encoding a recombinant human TGF-β2 monomer (or a fusion protein thereof).In some implementations, the composition comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0081] The pharma- ceutically acceptable carriers and excipients useful in this disclosure are conventional. For example, see Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, ed., Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st ed. (2005). For example, parenteral formulations usually contain injectable fluids that are pharma- ceutical and physiologically acceptable fluid vehicles (e.g., water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol, etc.). For solid compositions (e.g., powder, pill, tablet or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate). Excipients that can be included are, for example, other proteins, such as human serum albumin or plasma preparations.

[0082] For administration of cells, various aqueous carriers for introducing the cells can be used (e.g., buffered saline, etc.). These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharma- ceutically acceptable auxiliary substances as needed to approximate physiological conditions (e.g., pH adjusting and buffering agents, toxicity adjusting agents, etc. (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.)). The concentrations in these formulations can vary widely and are selected primarily based on fluid volume, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the subject.

[0083] The dosage form of the composition is determined by the mode of administration selected. For example, in addition to injectable fluids, topical, inhalant, oral and suppository formulations can be used. Topical preparations can include eye drops, ointments, sprays, patches, etc. Inhalant preparations can be liquid (e.g., solutions and suspensions), and can include mists, sprays, etc. Oral preparations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Suppository preparations can also be solid, gel, or in suspension form. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art.

[0084] Compositions (e.g., pharmaceutical compositions) containing recombinant human TGF-β2 monomer or fusion protein (or nucleic acid molecule / vector encoding TGF-β2 monomer or fusion protein) can be formulated in unit dosage forms suitable for individual administration of precise dosage amounts. The amount of TGF-β2 monomer, fusion protein, nucleic acid molecule or vector administered will depend on the subject being treated, the severity of the affliction, and the mode of administration, and is best left to the judgment of the prescribing clinician. Within these ranges, the formulation to be administered may contain a quantity of active component(s) sufficient to achieve the desired effect in the subject being treated.

[0085] The TGF-β2 monomers, or compositions thereof, can be administered to humans or other animals in various ways (e.g., topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation, or via suppository) to the tissues where they are effective. The particular mode of administration and dosing regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is preventative). Treatment may require daily or multiple daily administrations of the compound(s) for a period of several days to several months or even years.

[0086] V. Illustrative Provisions Clause 1. A recombinant transforming growth factor (TGF)-β2 monomer comprising: a cysteine ​​to serine or a cysteine ​​to arginine substitution at an amino acid residue corresponding to residue 77 of SEQ ID NO:1; a deletion of the α3 helix corresponding to amino acid residues 52-71 of SEQ ID NO:1; a lysine to arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO:1; a leucine to arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO:1; an alanine to lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO:1; a lysine to arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO:1; (i) an arginine to lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO:1; a valine to arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO:1; a leucine to valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO:1; an isoleucine to valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO:1; a threonine to lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO:1; and an isoleucine to valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO:1; (ii) a cysteine ​​to valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO:1; and a cysteine ​​to alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO:1; or (iii) a cysteine ​​to valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO:1; a cysteine ​​to alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO:1; an arginine to lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO:1; a valine to arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO:1; a leucine to valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO:1; an isoleucine to valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO:1; a threonine to lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO:1; and an isoleucine to valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO:1. 4. A recombinant TGF-β2 monomer comprising:

[0087] Clause 2. The recombinant TGF-β2 monomer of clause 1, comprising a cysteine ​​to arginine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1.

[0088] Clause 3. The recombinant TGF-β2 monomer according to clause 2, wherein the amino acid sequence of said TGF-β2 monomer comprises or consists of SEQ ID NO:4.

[0089] Clause 4. The recombinant TGF-β2 monomer according to clause 2, wherein the amino acid sequence of said TGF-β2 monomer comprises or consists of SEQ ID NO:6.

[0090] Clause 5. The recombinant TGF-β2 monomer of clause 1, comprising a cysteine ​​to serine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1.

[0091] Clause 6. A recombinant TGF-β2 monomer according to clause 5, wherein the amino acid sequence of said TGF-β2 monomer comprises or consists of SEQ ID NO:5.

[0092] Clause 7. A recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of said TGF-β2 monomer comprises or consists of SEQ ID NO:7.

[0093] Clause 8. A recombinant TGF-β2 monomer according to any one of clauses 1 to 7 which is PEGylated.

[0094] Clause 9. A recombinant TGF-β2 monomer according to any one of clauses 1 to 7 which is glycosylated or hyperglycosylated.

[0095] Clause 10. A recombinant TGF-β2 monomer according to any one of clauses 1 to 9, further comprising a radiotherapeutic agent, a cytotoxic agent for chemotherapy, a drug, an imaging agent, a fluorescent dye, or a fluorescent protein tag.

[0096] Clause 11. A fusion protein comprising a recombinant TGF-β2 monomer according to any one of clauses 1 to 10 and a heterologous protein.

[0097] Clause 12. The fusion protein of clause 11, wherein said heterologous protein comprises a protein tag, an Fc domain, albumin, an albumin binding polypeptide, an antibody, an antigen-binding fragment of an antibody or a targeting moiety.

[0098] Clause 13. A fusion protein according to clause 11 or clause 12, wherein the fusion protein is a single polypeptide chain.

[0099] Clause 14. A fusion protein according to clause 11 or clause 12, wherein the fusion protein forms a dimeric polypeptide.

[0100] Clause 15. The fusion protein of clause 11 or clause 12, wherein the fusion protein is a heterodimer.

[0101] Clause 16. A fusion protein according to clause 11 or clause 12, wherein the fusion protein is multimeric.

[0102] Clause 17. An isolated nucleic acid molecule encoding a recombinant TGF-β2 monomer according to any one of clauses 1 to 10 or a fusion protein according to any one of clauses 11 to 16.

[0103] Clause 18. The nucleic acid molecule of clause 17, operably linked to a promoter.

[0104] Clause 19. A vector comprising the nucleic acid molecule of clause 17 or clause 18.

[0105] Clause 20. An isolated cell comprising the nucleic acid molecule of clause 17 or clause 18, or the vector of clause 19.

[0106] Clause 21. The isolated cell of clause 20, wherein the cell is a T lymphocyte.

[0107] Article 22. a recombinant TGF-β2 monomer according to any one of clauses 1 to 10, a fusion protein according to any one of clauses 11 to 16, a nucleic acid molecule according to clause 17 or clause 18, a vector according to clause 19, or an isolated cell according to clause 20 or clause 21; and a pharma- ceutically acceptable carrier, diluent, or excipient; A composition comprising:

[0108] Clause 23. A method of inhibiting TGF-β signaling in a cell, comprising contacting the cell with an effective amount of a recombinant TGF-β2 monomer according to any one of clauses 1-10, a fusion protein according to any one of clauses 11-16, a nucleic acid molecule according to clause 17 or clause 18, a vector according to clause 19, an isolated cell according to clause 20 or clause 21, or a composition according to clause 22.

[0109] Clause 24. A method of inhibiting TGF-β signaling in a subject having a disease or disorder associated with aberrant TGF-β signaling comprising administering to the subject an effective amount of a recombinant TGF-β2 monomer of any one of clauses 1-10, a fusion protein of any one of clauses 11-16, a nucleic acid molecule of clause 17 or clause 18, a vector of clause 19, an isolated cell of clause 20 or clause 21, or a composition of clause 22.

[0110] Clause 25. A method of treating a disease or disorder associated with aberrant TGF-β signaling in a subject, comprising administering to the subject a therapeutically effective amount of a recombinant TGF-β2 monomer according to any one of clauses 1-10, a fusion protein according to any one of clauses 11-16, a nucleic acid molecule according to clause 17 or clause 18, a vector according to clause 19, an isolated cell according to clause 20 or clause 21, or a composition according to clause 22.

[0111] Clause 26. The method of clause 24 or clause 25, wherein said disease or disorder associated with aberrant TGF-β signaling is a fibrotic disorder.

[0112] Clause 27. The method of clause 24 or clause 25, wherein said disease or disorder associated with aberrant TGF-β signaling is breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, renal cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer or thyroid cancer.

[0113] Clause 28. The method of clause 24 or clause 25, wherein said disease or disorder associated with aberrant TGF-β signaling is an ocular disease.

[0114] Clause 29. The method of clause 24 or clause 25, wherein said disease or disorder associated with aberrant TGF-β signaling is a genetic disorder of connective tissue. EXAMPLES

[0115] Working Example Example 1: Modified TGF-β2 Monomer for In Vivo Administration This example describes studies evaluating modifications to enhance in vivo delivery of TGF-β2 monomer.

[0116] A TGF-β monomer engineered to prevent dimerization and thereby prevent binding and recruitment to the TGF-β type I receptor (TβRI) is described in WO 2018 / 094173, which is incorporated herein by reference in its entirety. The TGF-β2 monomer, mmTGF-β2-7M, contains a C77S substitution and a deletion of the α3 helix to prevent dimerization, and further contains seven amino acid substitutions that enhance high affinity TβRII binding and two substituted basic residues to increase its charge and thus its solubility (FIG. 1A). To improve the properties of this monomer, four variants of mmTGF-β2-7M were generated (SEQ ID NOs: 4-7). These are described in Table 1 and in FIGS. 1B-1E. The positions of the single amino acid substitutions and deletions are relative to human TGF-β2 as shown in SEQ ID NO: 1. Table 1. TGF-β monomer variants [Table 1-1] [Table 1-2]

[0117] As described in more detail below, substitution of C77 with arginine, together with substitution of V79 with arginine instead of serine as in mmTGF-β2-7M, allowed monomer formation and reduced aggregation of mmTGF-β2-7M2R (FIG. 1B). Reduction of misfolding was achieved by either C7V and C16A substitution (mmTGF-β2-2M-Del7-16; FIG. 1C), or by replacing the cystine knot region with that of PRDC (mmTGF-β2-7M-PRDC; FIG. 1D), by removal of the C7-C16 disulfide. The monomer, mmTGF-β2-7M2R-Del7-16 (FIG. 1E), contains modifications that reduce aggregation and improve folding. SEQ ID NO:1 - WT human TGF-β2 ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSKVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS SEQ ID NO:2 - mmTGF-β2 ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS SEQ ID NO:3 - mmTGF-β2-7M ALDAAYCFRNVQDNCCLRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS SEQ ID NO:4 - mmTGF-β2-7M2R ALDAAYCFRNVQDNCCLRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPRCRSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS SEQ ID NO:5 - mmTGF-β2-2M-Del7-16 ALDAAYVFRNVQDNCALRPLYIDFRRDLGWKWIHEPKGYNANFCAGACPYRASKSPSCVSQDLEPLTILYYIGRTPKIEQLSNMIVKSCKCS SEQ ID NO:6 - mmTGF-β2-7M2R-Del7-16 ALDAAYVFRNVQDNCALRPLYIDFRKDLGWKWIHEPKGYNANFCAGACPYRASKSPRCRSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCKCS SEQ ID NO:7 - mmTGF-β2-7M-PRDC KEVLASSQEALVVTERKYLKSDWCKLRPLYIDFRKDLGWKWIHEPKGYNANFCYGQCNSFYIPRHVKKEEDSFQSSAFCVSQDLEPLTIVYYVGRKPKVEQLSNMIVKSCRCMSV

[0118] In some implementations, any of the above sequences includes an N-terminal methionine (M) residue.

[0119] Elimination or reduction of tendency to clump Modifications that would eliminate or reduce the tendency of mmTGF-β2-7M to aggregate were first investigated. The engineered mmTGF-β2-7M monomers were previously shown to have a much lower tendency to aggregate than wild-type TGF-β2, but mmTGF-β2-7M nevertheless retains some tendency to aggregate (Kim et al., J Biol Chem 292(17):7173-7188, 2017). This was demonstrated by two-dimensional (2D, 2E) and (3D) aggregation when recorded either in the absence (Figure 2D, 2E) or in the presence (Figure 2F) of the non-denaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). 1 H- 15 The amide backbone, as detected by N NMR shift correlation (HSQC, heteronuclear single-quantum correlation) spectroscopy 1 H-15 This was evident from the appearance of the N signal. In the absence of CHAPS, the backbone amide signal was highly variable in intensity, and some were barely detectable, especially at pH 7.2, where the solubility of the protein is known to be reduced compared to that at pH 4.6. This type of variation in signal intensity is caused by the transient formation of higher order aggregates. The formation of such aggregates is related to the rotational correlation time (τ c ) and therefore broadens the NMR signal, decreasing signal intensity. It was observed that the addition of increasing concentrations of CHAPS at either pH 4.6 or 7.2 resulted in an improvement in the intensity of many signals and therefore an increase in their homogeneity in the observed spectrum (Figure 2F). The improvement in signal intensity was dependent on the concentration of CHAPS, with substantial improvement occurring up to a concentration of approximately 10 mM.

[0120] It was hypothesized that the role of CHAPS in reducing the aggregation of mmTGF-β2-7M was due to the transient formation of aggregates through several hydrophobic residues remaining in a region of the molecule best described as the base of the fingers that were part of the dimer interface in wild-type TGF-β2 homodimers (Figure 1A). Several substitutions were tested that were found to have little effect on aggregate formation, but substitution of two residues in mmTGF-β2-7M to arginines, S57R and V59R (Figure 1B), which correspond to the C77S and V79R substitutions for wild-type human TGF-β2 of SEQ ID NO: 1, significantly reduced the tendency to aggregate. This variant of mmTGF-β2-7M with the two residues replaced with arginines is referred to as mmTGF-β2-7M2R (SEQ ID NO: 4). Evidence for a reduced tendency to aggregate was the much more uniform NMR signal intensity observed for this variant, regardless of pH or whether non-denaturing CHAPS was added (Figures 2A-2C).

[0121] Modifications to improve folding TGF-β proteins are formed from monomers classified as having a cystine-knot growth factor fold (CKGF) (Hinck et al., Cold Spring Harb Prospect Biol 8(12):a022103, 2016). This fold is present in all proteins of the TGF-β family, but is also found in many other signaling proteins and signaling protein antagonists in humans. These include the signaling proteins platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and nerve growth factor (NGF) and antagonists such as noggin, sclerostin, and Dan and Cerberus related protein (PRDC). The TGF-β family of proteins is unique among the CKGF proteins in that they all have an N-terminal prodomain. Although the role of the prodomains is still being investigated, they are known to have a regulatory role for many proteins in the family (Hinck et al., Cold Spring Harb Prospect Biol 8(12):a022103, 2016). This regulation results from the binding of the prodomain to the growth factor domain (GFD), sometimes with sufficient (nanomolar to subnanomolar) affinity to completely block the ability of the GFD to bind type I and type II receptors. Some prodomains (e.g., those for TGF-β1, TGF-β2, and TGF-β3) not only bind the GFD with very high affinity, but also are required for the proper folding of the GFD, thus maintaining an inactive (latent) form until they are activated. The GFD of TGF-β, like that of other CKGF proteins, is characterized by a cystine knot, a structural motif stabilized by three disulfides (Schwarz, Biol Chem 398(12): 1295-1308, 2017). Because the three disulfides are very close to each other in space, their formation is complex, and in addition to the exact one, there are many possible alternative topological arrangements.

[0122] It is related to mmTGF-β2-7M because it retains a cystine knot (as well as one additional disulfide, known as the 8-17 disulfide (corresponding to the cysteines at residues 7 and 16 with respect to SEQ ID NO:1, and thus referred to herein as "7-16"). One method of producing the mmTGF-β2-7M protein is to express it in bacteria in the form of insoluble inclusion bodies and refold the protein to form the native pair of disulfides (Huang and Hinck, Methods Mol Biol 1344:63-92, 2016). Overall folding yields are nevertheless limited by misfolding and aggregates that form as a result of improper pairing of its eight cysteine ​​residues. mmTGF-β2-7M protein can also be produced by expressing the protein in a eukaryotic host as a secreted protein (but lacking the prodomain, unlike the wild-type TGF-β homodimer). However, attempts to use this method for expression of mmTGF-β2-7M have resulted in the formation of significant misfolded disulfide-linked aggregates.

[0123] Therefore, modifications aimed at improving the folding of mmTGF-β2-7M were investigated. To improve folding, each of the four disulfides of mmTGF-β2-7M was removed, one disulfide at a time. To do this, the two cysteines forming each disulfide were replaced with valine-alanine pairs, and the modified protein was then expressed, refolded, and purified according to previous procedures (Kim et al., J Biol Chem 292(17):7173-7188, 2017). To increase the chances of obtaining a natively folded protein, the substitutions were made in the context of the engineered TGF-β2 monomer, but with only two essential residues, K25 and K94, changed to those of TGF-β1, instead of the seven substitutions as in mmTGF-β2-7M. These variants were still predicted to bind TβRII with high affinity but fold with improved efficiency. This is because TGF-β2 is known to fold much more efficiently than TGF-β1 (Huang and Hinck, Methods Mol Biol 1344:63-92, 2016). The results showed that in this background, a variant in which the cysteines forming the 7-16 disulfide were replaced with valine and alanine (designated mmTGF-β2-2M-Del7-16 (SEQ ID NO: 5; Figure 1C)) folded natively (Figures 3A-3C), whereas variants in which the other three disulfides were removed (15-78, 44-109, and 48-111) were non-native. There was a strikingly significant variation in NMR signal intensity in the absence of CHAPS, suggestive of aggregation, but these disparities were lessened by the addition of CHAPS (Figures 3A-3C). The fact that the 7-16 disulfide can be removed without disrupting the folding of the protein indicates that this can result in significant improvements in folding whether the protein is produced in bacteria and refolded in vitro, or produced in eukaryotes as a secreted protein.

[0124] The third type of modification investigated was also aimed at improving the folding of mmTGF-β2-7M. The strategy chosen was to exploit the fact that there are some CKGF proteins (e.g., the bone morphogenetic protein (BMP) antagonist PRDC) that are naturally produced as monomers, do not have a prodomain, and do not rely on the prodomain for folding. To take advantage of the potential improvement in the folding of PRDC but retain the high affinity TβRII binding, a chimeric mmTGF-β2-7M:PRDC construct was generated in which the finger 1-2 and 3-4 regions of mmTGF-β2-7M (which are the regions responsible for binding TβRII) were grafted into the cystine knot region of PRDC. This construct, designated mmTGF-β2-7M-PRDC (SEQ ID NO: 7; FIG. 1D), was expressed in E. coli, refolded in a manner similar to that used for mmTGF-β27M (Kim et al., J Biol Chem 292(17):7173-7188, 2017), and purified to homogeneity using high-resolution cation exchange chromatography. Via NMR analysis, the protein was 1 It was shown to fold natively, as revealed by the dispersion of amide signals well outside the random coil region corresponding to 7.9–8.5 ppm in the H dimension (Figures 4A–4C), indicating that the cystine knot region of PRDC was well integrated with the finger region of mmTGF-β2-7M, making the design successful.

[0125] Binding properties of mmTGF-β2-7M variants A prerequisite for any designed mmTGF-β2-7M variant to be functional in cells and in vivo is that it binds TβRII with high affinity. Isothermal titration calorimetry (ITC) and native gels were used to evaluate the ability of the mmTGF-β2-7M variants described herein (mmTGF-β2-7M2R (SEQ ID NO: 4), mmTGF-β2-2M-Del7-16 (SEQ ID NO: 5), and mmTGF-β2-7M-PRDC (SEQ ID NO: 7)) to bind TβRII. ITC binding experiments were performed by injecting increasing amounts of TβRII into mmTGF-β2-7M2R (SEQ ID NO: 4) or mmTGF-β2-2M-Del7-16 (SEQ ID NO: 5) together with mmTGF-β2-7M (SEQ ID NO: 3) used as a reference control. These titrations produced readily detectable isotherms with large negative enthalpies and near 1:1 binding stoichiometries (Figure 5). Fits of the integrated heats to a 1:1 binding model revealed dissociation constants (K) for TβRII binding of 75.1 nM and 80.1 nM for mmTGF-β2-7M2R and mmTGF-β2-2M-Del7-16, respectively. D ) occurred (Figure 5). D is within experimental error of that determined for mmTGF-β2-7M (60.5 nM), indicating that substitutions introduced to reduce aggregation or improve folding did not have a detrimental effect on the ability of the protein to bind TβRII.

[0126] Binding of mmTGF-β2-7M-PRDC (SEQ ID NO: 7) was assessed instead using native gels. DAlthough this does not provide a quantifiable measure of binding, it does indicate high affinity binding, because detection of the complex requires that the two proteins remain bound for a time scale comparable to that of electrophoresis, which is approximately 1 hour. The native gel showed that mmTGF-β2-7M, mmTGF-β2-7M2R, and mmTGF-β2-7M-PRDC all formed bands migrating approximately ¼ of the length of the gel, while TβRII migrated for almost the entire length of the gel (Figure 4D). This, combined with the previous finding that mmTGF-β2-7M, mmTGF-β2-7M2R, and mmTGF-β2-7M-PRDC alone did not enter the gel, suggests that all three of these proteins bind TβRII with high affinity. This is consistent with the ITC results for the mmTGF-β2-7M and mmTGF-β2-7M2R variants, which we show also apply to mmTGF-β2-7M-PRDC.

[0127] Inhibitory properties of mmTGF-β2-7M variants To be functional in vivo, any designed mmTGF-β2-7M variant should inhibit TGF-β signaling in cells. To assess this for the disclosed mmTGF-β2-7M variants, mmTGF-β2-7M2R (SEQ ID NO: 4), mmTGF-β2-2M-Del7-16 (SEQ ID NO: 5), and mmTGF-β2-7M-PRDC (SEQ ID NO: 7), a HEK-293 TGF-β luciferase reporter cell line (where the cells are stably transfected with a TGF-β CAGA enhancer element fused to a luciferase reporter gene) was used. To assess inhibitory potential in this assay, the cells were plated in 96-well plates and various concentrations of mmTGF-β2-7M2R, mmTGF-β2-2M-Del7-16, and mmTGF-β2-7M-PRDC were added, with mmTGF-β2-7M used as a control. After 30 min, TGF-β signaling was stimulated by adding TGF-β3 to a final concentration of 10 pM, and after 12 h, the cells were lysed and luciferase activity was assessed. Results showed that mmTGF-β2-7M2R, mmTGF-β2-2M-Del7-16, and mmTGF-β2-7M-PRDCGAO each potently inhibited TGF-β3-induced signaling (fitted IC 50 values ​​of 53 nM, 111 nM, and 283 nM, respectively (Figures 6B-6D). The values ​​for both mmTGF-β2-7M2R and mmTGF-β2-2M-Del7-16 were within 2-fold of those measured for mmTGF-β2-7M (Figure 6A). This indicates that both of these proteins are roughly as effective as mmTGF-β2-7M (IC 50 58 nM). Although still potent, the IC 50 is 283 nM, which is approximately 5-fold reduced compared to mmTGF-β2-7M, indicating that mmTGF-β2-7M-PRDC is a functional TGF-β inhibitor, but its potency may be slightly compromised due to some small changes in the orientation of the two finger regions.

[0128] overview The mmTGF-β2-7M variants disclosed herein have substitutions that reduce their aggregation tendency and increase their folding tendency. Each of the above mmTGF-β2-7M variants has been shown to retain the ability to bind TβRII with high affinity and potently inhibit TGF-β3 signaling in cultured cells. Thus, the mmTGF-β2-7M variants of the present disclosure have attributes that improve their ability to be administered in vivo, thus providing new avenues for therapeutic intervention to attenuate the progression of TGF-β-mediated diseases.

[0129] In view of the many possible implementations to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated implementations are merely preferred examples of the invention and should not be construed as limiting the scope of the invention, which is rather defined by the following claims. The inventors therefore claim as their invention all that comes within the scope and spirit of these claims.

Claims

1. A recombinant transforming growth factor (TGF)-β2 monomer, a cysteine ​​to serine substitution or a cysteine ​​to arginine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:1; a deletion of the α3 helix corresponding to amino acid residues 52-71 of SEQ ID NO: 1; a lysine to arginine substitution at the amino acid residue corresponding to residue 25 of SEQ ID NO:1; a leucine to arginine substitution at the amino acid residue corresponding to residue 51 of SEQ ID NO:1; an alanine to lysine substitution at the amino acid residue corresponding to residue 74 of SEQ ID NO:1; a lysine to arginine substitution at the amino acid residue corresponding to residue 94 of SEQ ID NO:1; and (i) an arginine to lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO:1; a valine to arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO:1; a leucine to valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO:1; an isoleucine to valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO:1; a threonine to lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO:1; and an isoleucine to valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO:1; (ii) a cysteine ​​to valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO:1; and a cysteine ​​to alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO:1; or (iii) a cysteine ​​to valine substitution at the amino acid residue corresponding to residue 7 of SEQ ID NO:1; a cysteine ​​to alanine substitution at the amino acid residue corresponding to residue 16 of SEQ ID NO:1; an arginine to lysine substitution at the amino acid residue corresponding to residue 26 of SEQ ID NO:1; a valine to arginine substitution at the amino acid residue corresponding to residue 79 of SEQ ID NO:1; a leucine to valine substitution at the amino acid residue corresponding to residue 89 of SEQ ID NO:1; an isoleucine to valine substitution at the amino acid residue corresponding to residue 92 of SEQ ID NO:1; a threonine to lysine substitution at the amino acid residue corresponding to residue 95 of SEQ ID NO:1; and an isoleucine to valine substitution at the amino acid residue corresponding to residue 98 of SEQ ID NO:

1. A recombinant TGF-β2 monomer comprising:

2. 2. The recombinant TGF-β2 monomer of claim 1, comprising a cysteine ​​to arginine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:

1.

3. 3. The recombinant TGF-β2 monomer of claim 2, wherein the amino acid sequence of the TGF-β2 monomer comprises or consists of SEQ ID NO:

4.

4. 3. The recombinant TGF-β2 monomer of claim 2, wherein the amino acid sequence of the TGF-β2 monomer comprises or consists of SEQ ID NO:

6.

5. 2. The recombinant TGF-β2 monomer of claim 1, comprising a cysteine ​​to serine substitution at the amino acid residue corresponding to residue 77 of SEQ ID NO:

1.

6. 6. The recombinant TGF-β2 monomer of claim 5, wherein the amino acid sequence of the TGF-β2 monomer comprises or consists of SEQ ID NO:

5.

7. A recombinant transforming growth factor (TGF)-β2 monomer, wherein the amino acid sequence of said TGF-β2 monomer comprises or consists of SEQ ID NO:

7.

8. The recombinant TGF-β2 monomer of claim 1, which is PEGylated.

9. 2. The recombinant TGF-β2 monomer of claim 1, which is glycosylated or hyperglycosylated.

10. 10. The recombinant TGF-β2 monomer of claim 1, further comprising a radiotherapeutic agent, a cytotoxic agent for chemotherapy, a drug, an imaging agent, a fluorescent dye, or a fluorescent protein tag.

11. A fusion protein comprising the recombinant TGF-β2 monomer of claim 1 and a heterologous protein.

12. 12. The fusion protein of claim 11, wherein the heterologous protein comprises a protein tag, an Fc domain, albumin, an albumin-binding polypeptide, an antibody, an antigen-binding fragment of an antibody, or a targeting moiety.

13. The fusion protein of claim 11 , wherein the fusion protein is a single-chain polypeptide.

14. The fusion protein of claim 11 , wherein the fusion protein forms a dimeric polypeptide.

15. The fusion protein of claim 11 , wherein the fusion protein is a heterodimer.

16. The fusion protein of claim 11 , wherein the fusion protein is multimeric.

17. An isolated nucleic acid molecule encoding the recombinant TGF-β2 monomer of claim 1.

18. 18. The nucleic acid molecule of claim 17 , operably linked to a promoter.

19. A vector comprising the nucleic acid molecule of claim 18.

20. 20. An isolated cell comprising the vector of claim 19.

21. 21. The isolated cell of claim 20, wherein the cell is a T lymphocyte.

22. The recombinant TGF-β2 monomer of claim 1; and a pharmaceutically acceptable carrier, diluent, or excipient; A composition comprising:

23. 1. A composition for use in a method for inhibiting TGF-β signaling in a cell, comprising the recombinant TGF-β2 monomer of claim 1, the method comprising contacting the cell with an effective amount of the recombinant TGF-β2 monomer of claim 1.

24. 10. A composition for inhibiting TGF-β signaling in a subject having a disease or disorder associated with aberrant TGF-β signaling, comprising the recombinant TGF-β2 monomer of claim 1.

25. 10. A composition for treating a disease or disorder associated with aberrant TGF-β signaling in a subject, comprising the recombinant TGF-β2 monomer of claim 1.

26. 26. The composition of claim 25, wherein the disease or disorder associated with aberrant TGF-β signaling is a fibrotic disorder.

27. 26. The composition of claim 25, wherein the disease or disorder associated with aberrant TGF-β signaling is breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, kidney cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer or thyroid cancer.

28. 26. The composition of claim 25, wherein the disease or disorder associated with aberrant TGF-β signaling is an ocular disease.

29. 26. The composition of claim 25, wherein the disease or disorder associated with aberrant TGF-β signaling is a genetic disorder of connective tissue.

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